I propose to study the mechanism of prolactin action. Prolactin's primary action involves the development of the mammary gland and lactation. This lactogenic hormone, like insulin and growth hormone, does not act via cyclic nucleotides. Recently we have been able to identify a soluble mediator of prolactin action which specifically stimulates casein messenger RNA production from isolated mammary nuclei. This mediator has been partially characterized. It is a small peptide of 1000-2000 daltons, heat stable and inactivated by trypsin. A new assay to measure casein gene expression, involving the insertion of a complementary cDNA probe to rabbit Alpha- or Beta-casein in the plasmid pBR322 will be used. We have been able to measure the specific hybridization of casein mRNA to the cloned cDNA on nitrocellulose filters. We are developing a "dot hybridization" assay for the rapid detection of multiple samples. This assay will be of great value in the purification of the soluble PRL mediator, which will be undertaken. Batches of rabbit mammary membranes will be exposed to PRL and the resulting soluble fraction will be lyophilized and passed over a Sephadex G-10 column. Classical purification procedures (ion exchange HPLC, gel exclusion, isoelectric focusing, etc.), will be utilized to purify this peptide. Following purification, the amino acid sequence will be determined and analogues will be developed. The mechanisms involved in the release of the mediator from membranes, the role of phosphatidylinositol metabolism, as well as the interaction of the mediator with DNA and the process of turning on the prolactin sensitive genes will be studied. The mechanism of action of other hormones, such as insulin, growth factors and gonadotropins will be compared to that of prolactin. It is possible that soluble mediators are released by a number of hormones, even perhaps by those which have been shown to act via cyclic nucleotides.